The overall behavior of CHAV-Gth is similar to that previously reported for VSV-Gth. Particularly, CHAV-Gth pre-fusion trimer is not stable in solution and low-pH-induced membrane association of CHAV-Gth is reversible. Furthermore, CHAV-Gth was crystallized in its low pH post-fusion conformation and its structure was determined at 3.6Å resolution. An overall comparison of this structure with the previously reported VSV-Gth post-fusion conformation, shows a high structural similarity as expected from the comparison of primary structure. Among the three domains of G, the pleckstrin homology domain (PHD) appears to be the most divergent and the largest differences are confined to the secondary structure of the major antigenic site of rhabdoviruses. Finally, local differences indicate that CHAV has evolved alternate structural solutions in hinge regions between PH and fusion domains but also distinct pH sensitive switches. Globally the comparison between the post fusion conformation of CHAV and VSV-G highlights several features essential for the protein’s function. It also reveals the remarkable plasticity of G in terms of local structures.Chandipura virus (CHAV), a member of the vesiculovirus genus, is an emerging human pathogen. As for other rhabdoviruses, CHAV entry into susceptible cells is mediated by its single envelope glycoprotein G which is both involved in receptor recognition and fusion of viral and cellular membranes. Here, we have characterized the fusion properties of CHAV-G. As for vesicular stomatitis virus  G, fusion is triggered at low pH below 6.5. We have also analyzed the biochemical properties of a soluble form of CHAV-G ectodomain , is an emerging pathogen associated with deadly encephalitis, principally among children, in India. CHAV single envelope glycoprotein G is involved in two successive steps of virus entry: receptor recognition that allows endocytosis of the virion and fusion of viral and cellular membranes that allows release of the viral nucleocapsid into the cytoplasm for the subsequent steps of infection. As for other rhabdovirus, fusion is triggered at low pH and catalyzed by a conformational change of G from its pre-fusion toward its post-fusion conformation. We have determined the crystalline structure of CHAV-G in its post-fusion conformation. An overall comparison of this structure with the previously reported post-fusion conformation of vesicular stomatitis virus G reveals the selective pressure of the humoral immune response: The major antigenic site, located in the most exposed domain of the pre-fusion conformation of the glycoprotein, is also the most structurally divergent region of G. The structure also highlights the remarkable plasticity of G in terms of local structures particularly in some hinge regions and reveals that the vesiculovirus can be classified in two groups based on the nature of their pH sensitive switches which trigger the conformational change of G. Vesiculovirus) is an emerging human pathogen associated with deadly encephalitis, principally among children in the tropical areas of India. CHAV can be isolated from natural populations of Phlebotomine sandflies [IND). This inference is reasonable due to the conservation of the amino acid (aa) sequences of the proteins of the two viruses [Chandipura virus . G has a huge ectodomain (452 aa for CHAV-G) anchored in the lipid bilayer by a single transmembrane domain (TMD) which is located upstream a short C-terminal intra-viral segment. The crystal structures of both PRE and POST states of VSVIND-G ectodomain  have been previously reported [Mature glycoprotein G is about 500 aa long  in the We compared the fusion activity of both CHAV-G and VSV-G in a cell-cell fusion assay. For this, BSR cells were co-transfected with a plasmid encoding G (either VSV-G or CHAV-G) and rabies virus (RABV) phosphoprotein fused to the green fluorescent protein (P-GFP). P-GFP is unable to passively diffuse into the nucleus . This alIND-G. At pH 6.3, syncytia were smaller for both glycoproteins. At pH 6.5, no syncytia were detected for CHAV-G whereas small syncytia (less than 20 nuclei) were still observed for VSVIND-G up to pH 7.0  was purified by successive steps of ultracentrifugation and chromatography as previously described [We purified CHAV-G ectodomain from a recombinant VSV in which the G gene was replaced for that of CHAV (VSV/CHAV-G) . This chescribed .th on linear 5–20% (w/v) sucrose gradients at different pHs and compared it with that of VSVIND-Gth.In the case of VSV-G, the pre-fusion trimer, although detected at the viral surface , is not th in the sucrose gradients was similar to that of VSVIND-Gth . However, the pK of the transition  was slightly higher for CHAV-Gth (pH 6.7) than for VSV-Gth (pH 6.5).The overall behavior of CHAV-GVIND-Gth . At pH vth by liposome flotation assay. CHAV-Gth/liposome interaction was assessed over a broad pH-range, from pH 5.7 to pH 8.5  to avoid rosette formation via aggregation of the protein through its fusion loops. In these experiments, we could obtain CHAV-Gth crystals at pH 4.6 and determine the structure of the protein at 3.6 Å resolution by molecular replacement. Statistics of data collection and refinement are given in de novo after the first few rounds of refinement and rebuilding, and the final composite simulated annealing omit map confirms unambiguous placing of main chain and most side chains except in the distal parts of FD  out of 90 in CHAV-G are in structurally equivalent positions with VSV-G . The larIND-G in this region is the result of the selective pressure from the humoral response of the host immune system.VSV-G segments containing both helix D and helix E are the most exposed part of the glycoprotein in its PRE conformation at the viral surface and, in fact, this region constitutes the major antigenic site of several rhabdovirus glycoproteins –35. ThisIND-G lies in the hinge R2 and R3 connecting both PHD and FD  can be accommodated by very diverse amino acid sequences.Alignment of G aa sequences for 12 vesiculoviruses  reveals IND-G , which are brought close together in the POST six-helix bundle . The absence of this pH-sensitive switch seems to be compensated by CHAV-G’s residues D269 and E234 which form an intra-protomer hydrogen bond with H209 in their vicinity . This cl program  was usedRemarkably, alignment of vesiculoviruses G aa sequences  reveals This different switch of CHAV results in a tighter interaction between the base of the six-helix bundle and the PH domain . As a coIND-G and CHAV-G structures reveal that the domains do not evolve at the same rate and that the major actor on G evolution is the immune system. As a consequence, the most exposed domain in the PRE conformation (i.e. the PHD), which is the target of neutralizing antibodies, is also by far the most divergent domain among vesiculoviruses. This domain, located at the top of the PRE conformation, is also probably involved in receptor recognition. Therefore, it is probable that the humoral immune response is at the origin of receptor diversification among the rhabdovirus family.The comparison of VSVThe principal finding of this work is that, vesiculovirus G have diverged quite far in some respects but retained important functional and structural features. These features can be thus pointed at as essential for the protein’s function. As an example, the relative orientation of CD and PHD is different in CHAV-G and VSV-G POST protomers, but the relative orientation of PHD and FD remains the same despite the sequence and local structure divergence of segments R2 and R3 and the largely different sets of interactions between FD. This highlights the mechanistic importance of maintaining PHD-FD orientation. This also shows that the most important feature for segments R2 and R3 is their flexibility, but not their secondary structure. As a consequence, the structural constraints which exert on these segments are weak and R2 and R3 can tolerate quite different aa sequences. As another example, although pH sensitive switches are absolutely required for the correct function of G, those switches can be located in alternate places, as in the CHAV-G D269-E234-H209 cluster, the counterpart of which does not exist in VSV-G Figs.  and S5.In conclusion, the vesiculovirus glycoprotein, despite experiencing strong functional constraints, exhibits a remarkable plasticity in terms of sequence and even local structures. This is reminiscent of the remarkable structural conservation of the class II architecture despite the lack of any sequence similarity ,40. Thisth was purified as previously described [th) as assessed by mass spectrometry. After digestion, the virus was pelleted and the viral pellet was suspended in 50 mM HEPES pH 7.5 containing 2% glycerol. The suspension containing Gth was clarified by ultracentrifugation at 25000 rpm (SW35 rotor) for 1h on a 20% w/v sucrose cushion. Gth purification was achieved on a DEAE trisacryl column , followed by a size exclusion step using a Superdex S200 10/30 column . Fractions containing Gth were concentrated up to 10 mg/ml and stored at −80°C until use.CHAV-Gescribed . Brieflyescribed  was prodBSR cells plated on glass coverslips at 70% confluence were co-transfected with pCAGGS plasmids encoding VSV-G or CHAV-G, and P-GFP plasmid encoding the phosphoprotein of rabies virus fused to GFP . Twenty th protein was determined by sucrose gradient centrifugation. 100 μg of VSVIND-Gth or CHAV-Gth were incubated in 200 μl of buffer containing either 50 mM MES-NaOH (for pH 5.5 to 6.7) or 50 mM Tris-HCl (for pH 7 to 8.5) plus 100 mM NaCl at 37°C for 30 minutes. The protein solution was overlaid on a 5-to-20% w/v continuous sucrose gradient in 100 mM NaCl, 50 mM Tris-HCl or 50mM MES-NaOH at the required pHs. Following centrifugation at 35,000 rpm for 16 hours in an SW41 rotor, 12 equal fractions were collected manually from the bottom of the gradient. When required, the fractions were dissolved and reconcentrated to the original volume using ultrafiltration devices (30 kDa molecular weight cut off) to get rid of the excess of sucrose. 20 μl of each fraction was analyzed on 10% SDS-PAGE gels stained with Coomassie blue.The oligomeric state of the Gth at the desired pH during 30 minutes at 37°C . The lipid-protein mix was mixed with a 80% w/v sucrose solution so that final concentration of sucrose was around 65%. The mixture was overlaid with 2 mL of 50% w/v sucrose solution and 1.5 mL of 10% w/v sucrose solution, both solutions at the desired pH. Cushions were spun for 16 hr at 20°C in a SW55 Beckman rotor at 45,000 r.p.m. 20 μL of the top and bottom fraction were analyzed by SDS-PAGE. Gth in the top fraction was considered as liposome-bond, Gth in the bottom fraction was considered as not interacting with the membranes. For the reversibility experiment, lipid-protein mix was re-incubated at pH 8.5 after a first stage at pH 5.7.Membrane flotation assays were performed as previously described . Brieflyth (4 mg/ml) supplemented with 0.2% DDM with 1 μl of the reservoir solution  and equilibrated against 500 μl of reservoir solution.Crystals were obtained at 293 K by the hanging drop vapour diffusion method. Drops were prepared by mixing 1 μl of CHAV-GCrystals were harvested in a cryoprotecting solution containing reservoir solution supplemented with 30% glycerol and flash-cooled by plunging in liquid nitrogen. Diffraction data were collected at 100 K at PROXIMA 1 and ID-29 beam lines in SOLEIL and ESRF synchrotrons (France). Data for the best crystal were integrated and scaled using XDS/XSCALE . The cryhttp://www.pymol.org).The structure was solved by molecular replacement (MR) with the PHENIX package . The VSVS1 FigG is depicted by domains colored as indicated in (TIF)Click here for additional data file.S2 FigThe regions of lowest B factors are colored in blue.(TIF)Click here for additional data file.S3 FigOBS-FCALC map  and simulated-annealing composite omit map  are both contoured at 1σ. Omit map was calculated using phenix.refine [The molecular switch H209/E234/D269 and neighboring amino acids , the R2-R3 hinge region  and the interface between helix H and F  are represented as sticks. Protein domains and segments are colored as described in x.refine .(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 FigIND-G. Residues located in the fusion loops are indicated by pink stars below the sequences. Residues involved in the formation of the pH sensitive molecular switches are indicated by dots below the sequences . Vesicu(TIF)Click here for additional data file.


Plasmodium vivax is present in most countries where P. vivax infection is endemic, but the underlying molecular mechanisms responsible remain unknown. Increased expression of P. vivaxcrt-o (pvcrt-o) has been correlated with in vivo CQ resistance in an area with low-grade resistance. We assessed pvcrt-o expression in isolates from Papua (Indonesia), where P. vivax is highly CQ resistant. Ex vivo drug susceptibilities to CQ, amodiaquine, piperaquine, mefloquine, and artesunate were determined using a modified schizont maturation assay. Expression levels of pvcrt-o were measured using a novel real-time quantitative reverse transcription-PCR method. Large variations in pvcrt-o expression were observed across the 51 isolates evaluated, with the fold change in expression level ranging from 0.01 to 59 relative to that seen with the P. vivax β-tubulin gene and from 0.01 to 24 relative to that seen with the P. vivax aldolase gene. Expression was significantly higher in isolates with the majority of parasites at the ring stage of development  compared to those at the trophozoite stage . Twenty-nine isolates fulfilled the criteria for ex vivo drug susceptibility testing and showed high variability in CQ responses . After controlling for the parasite stage, we found that pvcrt-o expression levels did not correlate with the ex vivo response to CQ or with that to any of the other antimalarials tested. Our results highlight the importance of development-stage composition for measuring pvcrt-o expression and suggest that pvcrt-o transcription is not a primary determinant of ex vivo drug susceptibility. A comprehensive transcriptomic approach is warranted for an in-depth investigation of the role of gene expression levels and P. vivax drug resistance.Chloroquine (CQ)-resistant  Plasmodium vivax was first documented in 1989 in samples from the island of New Guinea  in w Guinea . Over th endemic , with sinagement –6. Surve unknown .P. vivax have focused primarily on genes known to play a role in drug resistance in P. falciparum, particularly P. vivaxcrt-o (pvcrt-o) and pvmdr1 (pvcrt-o single nucleotide polymorphisms (SNPs) and in vivo treatment outcome  and PvCRT allelic variants (pfcrt haplotypes CIET (Dd2) and SNNT (7G8) showed CQ hypersensitivity compared to the CMNK wild type (HB3), CQ sensitivity was highest in pvcrt-o wild-type transfected cells.Molecular studies of CQ-resistant d pvmdr1 , 9. Wher outcome , 11, CQ variants . In thosvariants . Whereaspvcrt-o in modifying the clinical response to CQ was reported in a patient with severe malaria who had a 20-fold increase in pvcrt-o expression compared to three patients with nonsevere P. vivax infection , an area with high levels of CQ-resistant P. vivax  hospital with uncomplicated od cells . The posex vivo susceptibility profiles of the antimalarials chloroquine (CQ), amodiaquine (AQ), mefloquine (MFQ), piperaquine (PIP), and artesunate (AS) were measured using a protocol modified from the World Health Organization (WHO) microtest as described previously  was derived using an inhibitory sigmoid Emax model.The eviously , 19. Ex croscopy , 20. In The iRBCs were mixed in a 1:10 volume ratio with TRIzol reagent  and stored at −80°C until use. Total RNA was extracted using a slightly modified version of the manufacturer's instructions in which the RNA precipitation step with isopropanol was extended overnight. RNA quality was assessed using the 260-nm/280-nm and 230-nm/280-nm ratios as determined on a NanoDrop UV spectrophotometer , with values between 1.8 and 2 deemed acceptable. DNA digestion was performed by applying Ambion DNase treatment (Invitrogen), and the concentration of RNA was measured using the Quant-iT RNA assay (Invitrogen).cDNA synthesis was performed using Superscript III enzyme (Invitrogen) and oligo(dT)s (Invitrogen). A final quantity of 100 ng of total RNA was used in the reverse-transcription reaction following the manufacturer's instructions and cDNA was stored at −20°C until further processing.P. vivax β-tubulin gene and pvcrt-o ; the primer pair described by Suwanarusk et al.   was used to amplify cDNA. Primer-BLAST software and NetPrimer software were used to design primers spanning exon-exon junctions of the k et al.  was usedF247063) . cDNA wa50 = 31.2 nM) with 82% of parasites at the ring stage of development and 18% of parasites at the trophozoite stage was used as the calibrator in all RT-qPCR runs. A threshold cycle (TC) value of 0.2 was applied to all the experiments. Intra-assay variation (repeatability) was evaluated by measuring the standard deviation (SD) of data from six replicates of the calibrator sample relative to the mean TC value. Interassay variation (reproducibility) of the FC values was calculated by means of the intraclass correlation coefficient, taking into account only isolates with results determined in at least three independent experiments.Samples were run in triplicate for each experiment and the final fold change (FC) of expression for a sample was derived from the results of at least two independent experiments. A CQ-sensitive isolate  and the limit of quantification (LOQ) of the assays were established by evaluating triplicates of 10 2-fold serial dilutions of cDNA. Melt curve analyses were performed at the end of each run to confirm the specificities in each reaction by heating from 55 to 99°C with a ramping rate of 1°C/5 s. A random set of amplicons was sent for dideoxy sequencing to Macrogen Inc.  to confirm correct target gene sequence amplification.E = 10(−1/slope)  , where Eequation  and was  formula , which aPlasmodium species confirmation was undertaken using a gDNA template and a modified version of the protocol of Padley et al. as described elsewhere  was extracted from approximately 100 μl iRBC aliquots using a QIAamp DNA minikit (Qiagen). lsewhere , 25. Infi et al. . The flut test on log-transformed data. For categorical variables, percentages and corresponding 95% confidence intervals (CI) were calculated by assuming a binomial distribution.Statistical analysis was undertaken using SPSS software . Spearman's rank correlation and the Kruskal-Wallis test were used for nonparametric analysis. Agreement of the FC expression estimates between the two housekeeping genes was calculated by paired Ethical approval for this study has been obtained from the Eijkman Institute Research Ethics Commission, Eijkman Institute for Molecular Biology, Jakarta, Indonesia, and the Human Research Ethics Committee of the Northern Territory (NT) Department of Health & Families and Menzies School of Health Research, Darwin, Australia.P. vivax field isolates were used for ex vivo drug susceptibility testing and molecular analysis. Baseline characteristics of these isolates are summarized in A total of 51 venous samples were collected, and the corresponding P. vivax infection was confirmed by PCR in all samples. Forty-three (84%) isolates could be genotyped successfully at all three STR loci, with 23 (53%) polyclonal infections identified. The remaining 20 (47%) isolates, including the CQ-sensitive calibrator, were from monoclonal infections. The allele frequencies for each marker are illustrated in Fig. S1 in the supplemental material.Monospecies pvcrt-o, 1.96  for the P. vivax β-tubulin gene, and 1.94  for the P. vivax aldolase gene. Sequencing of the amplicons confirmed the identity of each gene target and melt curve analyses produced single peaks for each gene. Whereas the LOD and LOQ of the P. vivax β-tubulin gene and the P. vivax aldolase gene were ≤0.2 ng cDNA, the lowest cDNA concentration tested in this study , the LOD of pvcrt-o was 6.2 ng  and the LOQ was 12.5 ng . Overall, the P. vivax aldolase gene assay exhibited lower TC values than the P. vivax β-tubulin gene and pvcrt-o assays, with the TC values being similar in the latter two assays.The mean assay efficiencies were 1.93  for TC values for the control sample across 14 experiments were 25.4  for pvcrt-o, 23.9  for the P. vivax β-tubulin gene, and 20.7  for the P. vivax aldolase gene. pvcrt-o expression could be derived in 102 (81.6%) of the 125 assays conducted. The intraclass correlation coefficient (ICC) of the pvcrt-o FC of expression was 0.969  relative to P. vivax β-tubulin gene expression and was 0.922  relative to P. vivax aldolase gene expression. The TC values for the P. vivax β-tubulin gene and the P. vivax aldolase gene were highly correlated  . For all three genes, TC values were negatively correlated with the baseline parasitemia  .The mean pvcrt-o expression was 1.3  relative to the expression of the P. vivax β-tubulin gene and 0.9  relative to the expression of the P. vivax aldolase gene. The two pvcrt-o FC estimates were highly correlated  .The geometric mean FC of < 0.001) , althougpvcrt-o expression and the proportion of ring-stage parasites . Comparing the pvcrt-o expression levels of isolates with a majority (>90%) of ring-stage parasites and isolates with a majority (>90%) of trophozoite-stage parasites, the mean expression levels were significantly higher in the group with a majority of ring-stage parasites . After classifying the 51 isolates according to the proportion of ring-stage parasites into groups with ≤10% (n = 13), 11% to 50% (n = 9), 51% to 90% (n = 7), and >90% (n = 22), a significant trend for increasing pvcrt-o expression levels with increasing proportion of ring-stage parasites was found, with the median FC of expression being 0.6 (interquartile range: 0.4 to 11.2) relative to P. vivax β-tubulin gene expression , 23 for piperaquine (15.5 [5.4 to 50.9] nM), 20 for mefloquine (13.4 [1.4 to 43.2] nM), 27 for amodiaquine (18.4 [1.9 to 43.6] nM), and 25 for artesunate (2.6 [0.2 to 13.8] nM)  or the P. vivax aldolase gene  .In total, 29 isolates fulfilled the criteria for 3.8] nM) . There w= 0.843) , and thi= 0.843)  or in thpfcrt is a key determinant of CQR in P. falciparum  expression ranging from 0.01 to 59 relative to the P. vivax β-tubulin gene and from 0.01 to 24 relative to the P. vivax aldolase gene.lciparum , but thepvcrt-o observed in our study most likely reflect differences in parasite stage composition in the isolates, with 3-fold-greater expression in isolates with a majority of ring-stage parasites than in those with a majority of trophozoite-stage parasites. However, the relative levels of expression of the housekeeping genes did not differ significantly with parasite stage, suggesting that the observations for pvcrt-o were determined predominantly by variations in pvcrt-o expression rather than by variations in expression of either of the housekeeping genes.The highly variable expression levels of P. vivax isolates from the Brazilian Amazon, with FC levels ranging from 0.3 to 189 relative to the expression of the P. vivax β-tubulin gene  and the maebl gene   . Our resfe cycle , 29. Howfe cycle , 31, dat limited .pvcrt-o expression and ex vivo susceptibility to CQ or any of the other standard antimalarials tested. Key differences between the two studies include different transmission intensities, differences in the drug susceptibility phenotypes and profiles, and different levels of parasite synchronicity. In the current study, the relationship between pvcrt-o expression levels and ex vivo drug response was assessed in highly synchronous isolates with proportions of rings between 78% and 100%, whereas in the Brazilian study, the proportion of ring-stage parasites in the isolates ranged from 29% to 88%. Furthermore, drug susceptibility was at a relatively low level in the Amazon, with treatment failure occurring in less than 12% of patients treated with CQ, whereas in Papua in 2007, 15% of patients had early treatment failure following CQ and more than 65% failed treatment by day 28  proportion of polyclonal P. vivax infections. However, after restricting data analysis to monoclonal infections, there was still no correlation between pvcrt-o expression levels and drug susceptibility or, indeed, a trend to significance.Polyclonal infections can also confound the interpretation of P. vivax β-tubulin gene and P. vivax aldolase gene, the two housekeeping genes used in the current study, were selected on the basis of available data in PlasmoDB, showing relatively stable expression of the P. vivax aldolase gene between hours 0 to 20 and of the P. vivax β-tubulin gene between hours 23 to 40 of the 48-hour life cycle (pvcrt-o expression estimates obtained using these two control genes highlights the need to test and evaluate multiple housekeeping genes in the early phase of development of assays of relative quantification in Plasmodium.Quantification of gene expression by RT-qPCR is reliant upon the choice of suitable housekeeping genes. The fe cycle . The staS. cerevisiae yeast expression models by Baro and colleagues showed that wild-type PvCRT is already as efficient for CQ efflux as PfCRT variants associated with a CQ resistance phenotype. As a consequence, additional mutations to increase the efficiency of transportation of CQ across the digestive vacuole membrane, in order to confer a CQ resistance phenotype, do not seem to be necessary. Together with the observations from the current study, there is growing evidence that pvcrt-o may have a role in mediating CQR mechanisms in P. vivax that is different from that of pfcrt in P. falciparum.Two studies have shown evidence for the involvement of the PvCRT protein in CQ transportation and accumulation , 13. Funpvcrt-o expression modified ex vivo susceptibility to CQ in isolates from an area endemic for high-grade CQ-resistant P. vivax strains. Our results suggest that parasite staging is an important confounding factor that needs to be addressed in quantifying gene transcription levels in clinical Plasmodium field isolates. However, these observations do not rule out a possible role of pvcrt-o in a multigenic modulation of P. vivax CQR. Transcriptome analyses are needed to shed light on the precise role of pvcrt-o, and other loci, in conferring CQR in P. vivax.In conclusion, we found no evidence that


Compared with GN-NH2 modified UMEA, the GN-COOH modified UMEA showed better electrochemical reduction to DO, owing to better dispersing and more active sites. The GN-COOH on UMEA was electroreduced to reduced GN-COOH (rGN-COOH) to increase the conductivity and the catalysis performance. Finally, the palladium nanoparticles/rGN-COOH composite was incorporated into DO microsensor for the detection of DO.This paper investigated two different modification methods of graphene (GN) on ultramicroelectrode array (UMEA) and applied the GN modified UMEA for the determination of dissolved oxygen (DO). The UMEAs were fabricated by Micro Electro-Mechanical System (MEMS) technique and the radius of each ultramicroelectrode is 10 μm. GN-NH The sensor can be utilized as an effective measuring tool for analysis and control of the DO level in the complex fresh water environments. Among the mentioned principles, the most commonly used DO sensors are typically a Clark-type cell, which contains an electrode system including a selective membrane. The response current of the DO sensor is the oxygen reduced current at the cathode after the oxygen diffusing through the membrane. DO sensors presently reported are relatively large devices [Dissolved oxygen (DO) is a vital parameter in water monitoring. A number of biological reaction and chemical reaction in water are influenced directly or indirectly by the quantity of DO. The concentration of DO in water below 2 mg/L for some hours can cause large fish kills . The detet al.  investig [et al.  investig [et al. ,8, galva [et al.  and pote [et al. . Martin  [et al.  investig devices ,8,10 whiThe outstanding characteristics of ultramicroelectrode array (UMEA) are resulted from the radial diffusion. When the center-to-center separation is large enough to avoid the “shielding” effect, the radial diffusion is dominant and the maximum current density is achieved . UMEA haet al. [et al. [Graphene (GN) has several advantages, such as huge surface area, high conductivity and fast electron transfer . These uet al.  investig [et al.  modified2 and GN-COOH were self-assembled on gold ultramicelectrode by covalently bonding of two thiols compounds (mercaptopropionic acid and mercaptoethylamine). The characteristics of GN-NH2 and GN-COOH modified UMEAs were investigated by electroreducing of DO. The GN-COOH modified UMEAs were further electroreduced and modified by palladium nanoparticles (PdNPs) for DO microsensor fabrication.In this paper, GN-NH2.2.1.2 and GN-COOH (carboxyl ratio >5.0 wt.%) were obtained from Nanjing XFNANO Materials Tech Co., China. The N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDC), N-Hydroxy-succinimide (NHS), palladium nitrate, mercaptopropionic acid and mercaptoethylamine were purchased from Sigma and Aldrich. Teflon membrane was purchased from Sartorius, Germany. All other chemicals were of analytical grade. Deionized water was used throughout the experiment. Different DO solutions were prepared by Na2SO3 solutions. DO concentration was detected by commercial DO meter . Scanning electron microscope (SEM) analysis was carried out on S-4800 field emission scanning electron microscope produced by Hitachi . Raman spectra were obtained on an AvaRaman-532TEC  employing a 532 nm laser beam. Electrochemical experiments were performed on Gamry Reference 600 electrochemical measurement system  with a three-electrode cell. The cell consisted of a modified UMEA working electrode, a commercial saturated Ag/AgCl (CRE) or on-chip Ag/AgCl electrode (ORE), and an on-chip Pt counter electrode. The reference electrode in the DO microsensor was ORE, and the others were CRE.GN-NH2.2.2/SiNx. The radius of disk-shaped ultramicroelectrode is 10 μm, and the interelectrode spacing is 200 μm. The active area of UMEA is 0.035 mm2. SU-8 with different height was fabricated to form the reference electrode pool and the electrolyte pool. The photo of the UMEA chip is shown in The UMEA chip was fabricated by MEMS technique on glass. The cross-sectional view of the UMEA chip is shown in 2.3.2CH2COOH) solution was added on the surface of UMEA for 12 h at 4 °C. The surface of the modified electrode was washed with ethanol and deionized water successively, and then dried in the air. Because of the strong Au-S bond, HSCH2CH2COOH molecular were adsorbed on the active sites of UMEA. Then a mixed solution of EDC and NHS was applied onto the surface of HSCH2CH2COOH modified UMEA for 15 min at 4 °C to active the carboxylic group. Subsequently, the solution of GN-NH2 was applied onto the above electrode for 5 h at 4 °C and washed with deionized water. This step makes the carboxyl groups on the UMEA bonded with the amino group of GN-NH2 to form GN-NH2/UMEA.Exactly 10 mM of mercaptopropionic acid (HSCH2CH2NH2) solution was added on the surface of UMEA for 12 h at 4 °C. A mixed solution of NHS and EDC was applied into the GN-COOH solution to active the carboxylic group. The process of the active carboxylic group is shown in 2CH2NH2 modified UMEA to form GN-COOH/UMEA. The scheme of the GN-COOH modified UMEA is shown in Exactly 10 mM of mercaptoethylamine  and air-saturated (Curves b and d) LiCl solution are shown in The further modification of reduced graphene oxide with metal NPs can increase its electrical conductivity to a large extent . The for3.4.vs. ORE). The responses of chronoamperometry were recorded at different concentration of DO in solution. The linear range of DO microsensor is shown in 5) and the current of 0 min (I0) is adopted as the current response. With the increasing concentration of DO from 0.9 to 7.2 mg/L, the response current was plotted against the DO concentration resulting in a straight line. The linear equation is ΔI (nA) = −1.026C (mg/L) + 11.2, with correlation coefficient of 0.988 and sensitivity of 1.026 nA/(mg/L). This behavior permits the use of this microsensor to monitor DO in solution.The DO microsensor characteristics were verified by chronoamperometry method at the potential of −0.4 V (4.2 and GN-COOH on UMEA were compared. The GN-COOH/UMEA showed better distribution and electroreduction ability. The GN-COOH was electroreduced to rGN-COOH to increase the electron transfer and the electroreduction activity. Then the PdNPs were electroreduce on rGN-COOH to further increase the electroreduction ability to DO. The PdNPs/rGN-COOH/UMEA as working electrode was constructed into DO micosensor. The microsensor showed a satisfying performance of DO and can be further used for detecting of DO in environmental water.Two different GN were modified on the UMEA by using self-assembling method. The surface morphology and the electroreduction ability of GN-NH


Plasmodium vivax has been reported from many endemic countries in the world. In Ethiopia, P. vivax accounts for 40% of all malaria cases and CQ is the first-line drug for vivax malaria. Mutations in multidrug resistance 1 (pvmdr-1) and K10 insertion in the pvcrt-o genes have been identified as possible molecular markers of CQ-resistance (CQR) in P. vivax. Despite reports of CQ treatment failures, no data are currently available on the prevalence of molecular markers of P. vivax resistance in Ethiopia. The objective of this study was to determine the prevalence of mutations in the pvmdr-1 and K10 insertion in the pvcrt-o genes.Evidence for decreasing chloroquine (CQ) efficacy against P. vivax clinical isolates were collected from West Arsi district in Ethiopia. Sequencing was used to analyse polymorphisms of the pvcrt-o and pvmdr-1 genes.A total of 36 pvmdr-1 fragment showed the presence of two non-synonymous mutations at positions 976 and 1076. The Y → F change at codon 976 (TAC → TTC) was observed in 21 (75%) of 28 the isolates while the F → L change (at codon 1076), which was due to a single mutation (TTT → CTT), was observed in 100% of the isolates. Of 33 samples successfully amplified for the pvcrt-o, the majority of the isolates (93.9%) were wild type, without K10 insertion.Sequencing results of the P. vivax was identified. The fact that CQ is still the first-line treatment for vivax malaria, the significance of mutations in the pvcrt-o and pvmdr-1 genes and the clinical response of the patients’ to CQ treatment and whether thus an association exists between point mutations of the candidate genes and CQR requires further research in Ethiopia.High prevalence of mutations in candidate genes conferring CQR in  Plasmodium species infecting humans, Plasmodium vivax is the most widely distributed species and the cause of 25-40% of malaria cases worldwide [P. vivax malaria has received little attention and limited funds for research and control, since it usually produces less severe symptoms than falciparum malaria [P. vivax particularly to the only class of compounds available for killing the dormant liver stage is alarming and of high priority for research [P. falciparum resulting in increased morbidity and mortality. If a repetition is to be avoided, the threat of emerging CQ-resistant P. vivax needs to be acknowledged quickly and widely and substantial resources need to be allocated to validate and standardize tools necessary for characterization of drug-resistant P. vivax [P. vivax CQR has occurred in at least three Latin American countries  [P. vivax asexual blood stages than CQ (25 mg/kg over 3 days) co-administered with PQ (15 mg of PQ base/day for 14 days) over 28 days of follow-up [P. vivax resistance to CQ and the prospects for developing and standardizing reliable molecular markers of drug resistance, Goncalves et al. [in vivo and in vitro studies.Of the five orldwide , and sub malaria . Currentresearch -10. It iP. vivax . Further Brazil) . The fouollow-up . To unves et al.  reviewedP. falciparum, the molecular mechanism of P. vivax CQR remains elusive [P. falciparum have failed to demonstrate a strong correlation between pvcrt-o and pvmdr-1 genotypes and the CQR phenotype in P. vivax. Melo et al. [pvcrt-o and pvmdr-1 with CQR and severe P. vivax malaria, because parasites from patients with CQR presented up to 6.1-fold and 2.4-fold increase in pvcrt-o and pvmdr-1 expression levels, respectively, compared to the susceptible group in the Brazilian Amazon.Unlike in  elusive . This iso et al. , on the P. vivax is becoming more widespread, hindering management of clinical cases and posing a huge threat to the health of millions of people exposed to the risk of vivax malaria. Analysis of the single nucleotide polymorphisms (SNPs) in drug resistant genes has proved to be useful and important in monitoring drug resistance in malaria endemic countries [pvmdr-1) and K10 insertion in the pvcrt-o genes have been identified as possible molecular markers of CQR in P. vivax [pvcrt-o and pvmdr-1 genes and CQR [pvcrt-o and pvmdr-1 polymorphisms and CQR. Some reports suggest the Y976F mutation in pvmdr-1 to be associated with an increase in CQ IC50 value of P. vivax isolates in vitro [pvmdr-1 have been reported to have correlation with CQR although much work remains to link these mutations irrefutably with CQR [pvmdr-1 gene suggested reduced susceptibility to CQ [pvcrt-o in transgenic lines of P. falciparum modulates CQ response [et al. [pvcrt-o and pvmdr-1, respectively, in severe vivax malaria cases compared to isolates from non-severe vivax malaria patients. Another study in India showed the predominance of the wild-type pvmdr-1 and pvcrt-o alleles [pvmdr-1 gene, which could suggest the beginning of a trend towards decreased CQ sensitivity. In Thailand and Indonesia, where CQR is common, the pvmdr-1 (Y976F and F1076L) polymorphisms were also identified in P. vivax samples [P. vivax CQR remains relatively uncommon, the Y976F and F1076L polymorphisms are relatively infrequent [Drug resistance in ountries . MutatioP. vivax ,17. Few  and CQR . Neverthin vitro . Non-synwith CQR ,18,20. T [et al.  demonstr alleles  although samples . In Latifrequent ,24.P. vivax and artemether-lumefantrine (AL) for P. falciparum. Unlike in many malaria endemic countries in Africa, both P. falciparum and P. vivax substantially contribute to malaria morbidity in Ethiopia in relative proportions of 60 and 40%, respectively [pvmdr-1 and pvcrt-o genes are limited in Ethiopia. The study was, therefore, initiated to determine the SNPs in the pvmdr-1 and pvcrt-o genes.Presently, Ethiopia maintains a species-specific treatment policy: CQ without PQ is the first-line treatment for ectively ,26. In 1ectively  althoughectively -30. IndePlasmodium were recorded. Finger-prick blood samples were collected from patients and used for thick and thin blood film preparation. Slides were considered negative after examination of 100 high-power fields. Patients showing positive results for P. vivax infection were treated with CQ. Blood samples spotted on filter paper were used for molecular analyses.The samples for this study were collected from West Arsi district from November to December 2012. Malaria transmission is seasonal and unstable in this area. Study participants were patients seeking malaria diagnosis at the Aje Health Centre, located at 0382146.3 E, 071734.2 N and 1,852 m above sea level. Malaria diagnosis was confirmed by microscopy of Giemsa-stained blood films and the species of pvcrt-o (K10 insertion) and pvmdr-1 (Y976F and F1076L) genes were amplified by nested PCR using gene-specific primers .DNA was extracted from blood spots on filter paper using Chelex extraction methods as described elsewhere . The pvcStudy protocol was reviewed and approved by Institutional Review Boards of Aklilu Lemma Institute of Pathobiology, Addis Ababa University and of the Armauer Hansen Research Institute as well as the National Research Ethics Review Committee.Since this study was a preliminary exploratory study, a power calculation of sample size was not done. Data were entered, validated and analysed in Microsoft Excel 2010. Allele proportions were calculated for codons of interest by dividing the number of samples with a particular allele to the number of samples with an identifiable allele at that position.pvmdr-1 gene was successfully amplified and sequenced in 78% (28/36) of the P. vivax isolates. Two pvmdr-1 mutant alleles were identified: Y976F alone and Y976F-F1076L. The prevalence of pvmdr-1 Y976F mutation was 75% (21/28) (Table pvmdr-1 F1076L mutation.The d in 78% 8/36 of tpvcrt-o gene was successfully sequenced in 92% (33/36) of the isolates. Of the 33 samples successfully amplified for the pvcrt-o, the majority of the samples (93.9%) were wild type, without K10 insertion .CQ continues to be used for the treatment of  country -30,32. IP. vivax  because ty to CQ . In the ty to CQ . Unlike  [et al.  found thP. vivax isolates had the Y976F mutation in pvmdr-1. Sequencing results of the pvmdr-1 fragment showed the presence of two non-synonymous mutations at positions 976 and 1076. The Y → F change at codon 976 (TAC → TTC) was observed in 26 (75%) of 28 isolates. The second F → L change (at codon 1076), which was due to a single mutation (TTT → CTT), was observed in 100% of isolates. Whether isolates carrying the pvmdr-1 Y976F mutation responded to CQ treatments differently from those isolates with the wild-type sequence necessitates further in vivo therapeutic efficacy study in Ethiopia, but reports from Indonesia and Thailand suggest this to be the case [pvmdr-1 Y976F in areas where CQR P. vivax prevails versus CQ remain efficacious may indicate the correlation between CQR and sequence polymorphisms in pvmdr-1. In Papua Indonesia, where CQR P. vivax is present at high prevalence (>65%) and high level [pvmdr-1 Y976F mutation was present in all patients presenting to a clinic. In contrast, the sequence polymorphism in pvmdr-1 conferring Y976F was identified in only 25% of Thai isolates from an area where CQ remains efficacious. Ninety-six percent of Indonesian isolates  had Y976F mutation, compared to 25% of Thai isolates where CQ sensitivity was almost uniform [et al. [In the present study, 75% of the the case . The difgh level , the pvm uniform . The fac [et al.  the F107P. vivax has been reported from various studies. Given the high prevalence of the Y976F mutations in pvmdr-1 in Southeast Asia where CQR prevails [pvmdr-1 Y976F mutations identified in this study may be associated with the CQ treatment failure reported in Ethiopia. But the exact role of this mutation needs to be determined by a combination of in vitro and clinical observation studies in this country. The fact that all isolates carried the pvmdr-1 F1076L mutation, substitution in this codon may be less involved in the modulation of P. vivax susceptibility to CQ than the pvmdr-1 Y976F mutation given that CQ is still effective and widely used in Ethiopia. Indeed, the presence of pvmdr-1 F1076L mutation in all susceptible and mutant isolates challenged the role of pvmdr-1 polymorphisms in modulating CQ responses in P. vivax [pvmdr-1 polymorphisms have been recently suggested to be associated with CQR in Southeast Asia [pvcrt-o that have not been associated with CQR in P. vivax. The limitation of this study was that it did not determine drug resistance phenotype (either in vivo or in vitro) for the isolates undergoing molecular characterization at the pvmdr-1 and pvcrt-o genes. Indeed, withdrawal of a given drug is recommended when 10% of infections are not responding to treatment, although in practice, governments of poor countries leave it longer [P. vivax has great public health significance.In Ethiopia, CQR prevails , the higP. vivax . On the ast Asia  unlike pt longer . The facP. vivax accounts for about 40% of malaria cases, little attention has been given to the urgent public health need to detect and to closely monitor the progression of CQ-resistant vivax malaria in the country. This study has observed a high prevalence of the pvmdr-1 976 F allele, which is believed to be associated with CQR in P. vivax. In view of reports from elsewhere, the high prevalence of pvmdr-1 Y976F mutation identified in this study may be associated with the reported CQ treatment failure Ethiopia. However, determination of the exact role of this particular mutation in P. vivax CQ responses, as well as the roles of other identified pvmdr-1 and pvcrt-o gene mutations needs further research, involving a combination of in vitro and clinical observation studies in this country.Despite the fact that


The aim of the present study was to investigate the effect of the ultrasound-targeted microbubble destruction mediated (UTMD) herpes simplex virus-thymidine kinase (HSV-TK) and ganciclovir (GCV) system on ovarian cancer (OC). This study was conducted between June and December 2012 in the Animal Biosafety Level III Laboratory of Wuhan University. Mice with OC were randomly divided into four groups: i) HSV-TK plus microbubbles (MBs) plus ultrasound (US) (n=15); ii) HSV-TK plus US (n=15); iii) HSV-TK (n=15); and iv) phosphate-buffered saline (n=15). The inhibitory effect and survival time in the experimental groups were compared with those in the control group. The TK protein expression was detected by western blot analysis. Tumor cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and caspase-3 activity analysis. The data showed that the efficiency of HSV-TK gene transfection and the tumor inhibitory effects were significantly increased in the HSV-TK plus MBs plus US group compared with those in the control group (P<0.01). UTMD-mediated HSV-TK treatment has also improved the rat survival rate (P<0.01). In conclusion, UTMD can effectively transfect the HSV-TK gene into target tissues and exert a significant inhibitory effect on OC in mice. Ovarian cancer (OC) is the most common malignant tumor involving the ovaries and remains associated with a high mortality rate. Every year, ~191,000 cases are diagnosed worldwide . Thus, tin vitro and in vivo to treat diabetes, cardiovascular disease, lung cancer, thyroid cancer and even tendon diseases in experimental animal models  and spread on a lysogeny broth agar plate for 16 h of culture. The sequence was confirmed by the Beijing Genomics Institute . The positive clones were then selected for plasmid extraction. The SonoVue® MB contrast agent SF6 was purchased from Bracco ; 90% of the MB diameters were measured to be <6 μm. The average diameter of the MBs was 2.5 μm. The gene-loaded lipid MBs were prepared as described in the study by Wang et al  was used in a polymerase chain reaction amplification with upstream (GAATTCATGGCCTCGTACCCCGGC) and downstream (CTCGAGTCAGTTAGCCTCCCCCATC) HSV-TK primers to obtain ~1.2 kb of the target HSV-TK fragment. The HSV-TK target gene was then connected to a pMD-18T simple vector  to obtain the recombinant plasmid pMD-18T-TK. The recombinant plasmid was transformed into DH5a ng et al . The MBs6 cells/site OC cells  subcutaneously into the right armpit under isofluorane anesthesia. In this model, tumors typically grew to 0.2 cm3 by the 15th to 20th day after injection. Sixty mice were then selected and randomly divided into four groups: i) HSV-TK plus MBs plus US (n=15); ii) HSV-TK plus US (n=15); iii) HSV-TK (n=15); and iv) PBS (n=15). Ten mice in each group were selected for a 14-day survival rate observation.The animal experiments were performed in the Center for Animal Experiments/Animal Biosafety Level III Laboratory of Wuhan University , and were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Wuhan University. Eighty female BALB/c-nu mice, aged six weeks and with a body weight of 14–16 g, were purchased from the Hubei Center for Disease Control and Prevention . Mice were raised in specific-pathogen free rooms under controlled conditions with a temperature of 23±3°C and relative humidity of 50±20%. Each BALB/c-nu mouse was inoculated with 2×102. The pulse irradiation method was used for 5 min, with an interval time of 10 sec, according to the method described in the study by Zhou et al  was administered in the PBS group and HSV-TK  was administered in the other three groups. A US transducer  was applied on the HSV-TK plus MBs plus US and HSV-TK plus US groups for irradiation following the gene injection. The US probe was tightly placed on the tumor mass to ensure that the whole tumor was exposed to the US. The US frequency was 1 MHz and sound intensity was 2 W/cmou et al  Fig. 1)2. The pu3)=(a × b2)/2, where ‘a’ refers to the maximum diameter (mm) and ‘b’ refers to the minimum diameter (mm); and IR = (average tumor size in the control group - mean tumor volume in the treatment group)/mean tumor volume in the control group × 100%. The growth curve was drawn according to the size of the tumor volume. The weight of the mice was also noted for the evaluation of their condition.The maximum and minimum diameters of the tumor mass were measured with a digital caliper every two days. The size of the tumors was measured twice using the same method by two different experimenters. Tumor volume and inhibition rate (IR) were calculated on day 14 using the following formulae: Tumor volume . Briefly, the homogenates of the liver tissues were centrifuged, and protein (1 g) was incubated with Asp-Glu-Val-Asp-p-nitroanilide and reaction buffer for 90 min at 37°C. Absorbance measured at 405 nm was representative of caspase-3 activity. A terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was carried out using a commercial kit . Apoptotic cells were identified by a brown stain over the nucleus. A total of ~200 cells were counted per field, five fields were examined per slide and five slides were examined per group. The apoptotic index (AI) was calculated as follows: AI =  × 100%.Proteins were extracted using a protein extraction reagent (Sigma), following the manufacturer’s instructions. The TK protein was detected by western blotting. Concentrated gel (40 ml/l), separation gel (100 ml/l), pre-stained protein marker (3.0 μl) and sample total proteins (50 μg/hole) were prepared. The samples were added into 100 ml/l SDS for SDS-PAGE. The gel was removed when the bromophenol blue ran to the bottom, and the protein was synchronously transferred to the polyvinylidene difluoride membrane at 20 V for 50 min. This was then sealed for 4 h with 50 ml/l skimmed milk and Tris-buffered saline with Tween 20 (TBST) at room temperature (RT) following the trans-membrane procedure. The primary antibody was subsequently added  prior to incubation for 2 h at RT and maintenance overnight at 4°C. TBST was used to wash the membrane four times for 10 min/time. The horseradish peroxidase-conjugated secondary antibody  was then added for incubation, followed by agitation at RT for 2 h, washing of the membrane, imaging and exposure. The protein bands were normalized to GAPDH, and all blots were quantified with Quantity One software .Experimental data are presented as the mean ± standard deviation. The data were processed by the statistical analysis software SPSS version 16.0 . The analysis of variance was used to assess the IR. The Kaplan-Meier method was applied for the survival analysis. P<0.05 was considered to indicate a statistically significant difference.As the tumor size increased, the mice exhibited evident appetite loss, activity reduction, body weight loss and humpback posture. From the tumor growth curve, it was observed that the tumor growth in the HSV-TK plus MBs plus US group slowed significantly. Compared with the tumor sizes of the PBS group, the tumor sizes of the HSV-TK plus MBs plus US group were significantly lower during the last four days of observation (P<0.01) . The tumTUNEL staining was performed to detect tumor cell apoptosis in each group. The TUNEL-positive cells (apoptotic cells) were stained brown. The tumor cells in each group underwent apoptosis to different degrees. The tumor cell apoptosis in the HSV-TK plus MBs plus US group was the most evident. The AI was highest (42.20±5.68%) in the HSV-TK plus MBs plus US group, which was significantly higher than that in the HSV-TK plus US , HSV-TK  and PBS  groups . In addiThe TK protein expression was detected in tumor tissues by western blot analysis. It was observed that a single band appeared in each group at 25 kd. The band in the HSV-TK plus MBs plus US group was the most evident , characterized by a higher band intensity (P<0.01) . The banOC is the leading cause of mortality from gynecological malignancies. Despite the fact that a response to initial therapy is exhibited by the majority of patients presenting with advanced disease, only 10–15% maintain a complete response following first-line therapy . Thus, uIn the present study, western blot analysis was performed to confirm the TK expression, and it was revealed that UTMD could directly deliver the TK suicide gene to OC cells in this mouse model. The results of the western blot analysis showed that there was no evident TK protein expression in the major organs, including the heart, lung, liver and kidneys, which meant that the transfection of the TK gene was confined within the tumor cells. The mechanisms underlying UTMD-mediated gene transfer remain incompletely understood. The liposome MB is composed of polyethylene glycol and liposome, and the mean diameter of the liposome MB is smaller than that of red blood cells or conventional MBs; thus the liposome MB could be developed as a gene transfection tool and delivered into specific tissues . MB disrA clear inhibitory effect on OC was observed in the present study. Compared with the other groups, tumor cell apoptosis in the HSV-TK plus MBs plus US group was more apparent. In addition, the tumor growth was significantly inhibited in the HSV-TK plus MBs plus US group. These findings established proof of concept that UTMD can impact tumor biology. Furthermore, it was observed that mice in the HSV-TK + MBs + US treatment group showed improved appetite, enhanced activity and lower body weight loss during this study. HSV-TK gene delivery into tumor cells followed by treatment with the antiviral drug GCV is the most common experimental and clinical model for gene therapy. The TK metabolizes the GCV into a monophosphorylated form, which is subsequently converted to a toxic triphosphate molecule by other cellular kinases. This toxic GCV metabolite is then incorporated into the DNA of replicating cells, leading to premature strand termination, replication failure and cell death. This type of pro-drug activation is widely described as a ‘suicide’ gene therapy ,23. As oIn conclusion, UTMD was used as a gene delivery method in the treatment of mouse OC by HSV-TK/GCV. The anti-tumor efficacy of HSV-TK and the mouse survival rate were markedly improved following use of the UTMD-mediated HSK-TK/GCV system. The UTMD technology is expected to provide a novel strategy for targeted OC therapy.


Millions of HIV-infected Africans are living longer due to long-term antiretroviral therapy (ART), yet little is known about glucose metabolism disorders in this group. We aimed to compare the prevalence of glucose metabolism disorders among HIV-infected adults on long-term ART to ART-naïve adults and HIV-negative controls, hypothesizing that the odds of glucose metabolism disorders would be 2-fold greater even after adjusting for possible confounders.In this cross-sectional study conducted between October 2012 and April 2013, consecutive adults (>18 years) attending an HIV clinic in Tanzania were enrolled in 3 groups: 153 HIV-negative controls, 151 HIV-infected, ART-naïve, and 150 HIV-infected on ART for ≥ 2 years. The primary outcome was the prevalence of glucose metabolism disorders as determined by oral glucose tolerance testing. We compared glucose metabolism disorder prevalence between each HIV group vs. the control group by Fisher’s exact test and used multivariable logistic regression to determine factors associated with glucose metabolism disorders.HIV-infected adults on ART had a higher prevalence of glucose metabolism disorders (49/150 (32.7%) vs.11/153 (7.2%), p<0.001) and frank diabetes mellitus (27/150 (18.0%) vs. 8/153 (5.2%), p = 0.001) than HIV-negative adults, which remained highly significant even after adjusting for age, gender, adiposity and socioeconomic status (OR = 5.72 (2.78–11.77), p<0.001). Glucose metabolism disorders were significantly associated with higher CD4+ T-cell counts. Awareness of diabetes mellitus was <25%.HIV-infected adults on long-term ART had 5-fold greater odds of glucose metabolism disorders than HIV-negative controls but were rarely aware of their diagnosis. Intensive glucose metabolism disorder screening and education are needed in HIV clinics in sub-Saharan Africa. Further research should determine how glucose metabolism disorders might be related to immune reconstitution. We hypothesized that the prevalence of GMDs would be at least two times greater among HIV-infected adults on ART for ≥2 years compared with HIV-negative controls and that this difference would not be explained by differences in age, gender or adiposity between groups.This was a cross-sectional study designed to compare the prevalence of GMDs among 3 groups: 1) HIV-infected, ART-naïve adults, 2) HIV-infected adults on ART for ≥2 years and 3) HIV-negative controls.The study was conducted in the outpatient HIV clinic of Bugando Medical Centre (BMC) in Mwanza, Tanzania. BMC is the zonal hospital for the Lake Victoria Zone in northwest Tanzania, serving a population of approximately 13 million. The HIV prevalence in the Lake Zone is 6% , similarHIV-negative adult treatment partners (control group),HIV-infected adults newly establishing care in last 3 months, not yet on ART  &HIV-infected adults on ART for ≥2 years .All of the study population was recruited from the BMC HIV clinic. The study included 3 groups of adults  for the purpose of comparisons:HIV-negative adult treatment partners were chosen as a control group in order to provide a control population with similar socioeconomic status to the 2 groups of HIV-infected adults. All treatment partners who attended the BMC HIV clinic during the study period were eligible for enrolment. Treatment partners were not matched to the HIV-infected adults who were enrolled. Exclusion criteria included pregnancy and failure to attend a follow-up visit on the day after enrolment.On the day of enrolment, a revised version of the WHO STEPs questionnaire was administered to determine the prevalence of glucose metabolism disorders and its risk factors . The WHOAt enrolment, blood samples were obtained and the CD4 count was measured using an automated BD FACS Calibur Machine . A urine pregnancy test was performed on women whose last menstrual period was >1 month prior to the date of interview. Enrolled study subjects were instructed to return to the clinic the following morning after an overnight fast.All participants underwent an oral glucose tolerance test (OGTT) according to WHO protocol . After aGMD was as defined as the presence of impaired fasting blood glucose, impaired glucose tolerance, or diabetes mellitus, according to the WHO definitions . ImpaireHypertension was defined as a sustained elevation of systolic blood pressure (SBP) ≥140 and/or diastolic blood pressure (DBP) ≥90 on 2 different days or current antihypertensive therapy according to the Joint National Committee 7 (JNC-7) definition, and graded according to the same guidelines . CentralThe primary outcome of the study was the prevalence of GMDs (as defined above). The primary study analysis was to compare the prevalence of GMDs between each HIV-infected group and the HIV-negative control group. According to a recent population-based survey, 1.9%  of adults in Mwanza city have diabetes mellitus while 16.4%  have hypertension . We hypothesized that 10% of HIV-infected adults on ART would have glucose metabolism disorders. Using Fisher’s exact test, we calculated that 150 patients in each group would provide 90% power to detect this difference for p<0.05.Data analysis was done using STATA version 11 . Descriptive statistics were computed by determining medians (interquartile ranges) for continuous variables and proportions (percentages) for categorical variables. Differences between medians were determined using the rank sum test and differences between proportions were determined using Fisher’s exact test. For ordered categorical variables, the nonparametric test for trend was used. P-values of less than 0.05 were considered significant.Factors associated with GMDs were determined by logistic regression. Multiple logistic regression models were performed in order to determine whether the relationship between HIV status and GMD could be explained by confounding. All baseline characteristics were evaluated by a pre-determined, minimally-adjusted logistic regression model adjusting for age and sex, since these were expected to differ between groups. Furthermore, additional, pre-determined multivariable analyses were performed to adjust for BMI and waist/hip ratio  as well as a fully-adjusted model including all variables with a p-value of <0.05 by minimally adjusted multivariable analysis. BMI and waist-to-hip ratio were not included together in any models due to collinearity. Variables associated with HIV infection and ART use  were not included in the multivariable models due to collinearity with the group variables and the smaller number of subjects with these additional variables. For associated factors, odds ratios (OR) were determined with 95% confidence intervals (95% CI). The likelihood ratio test was used to compare the logistic regression models.The study was approved by the Institutional Review Boards at Bugando Medical Centre and Weill Cornell Medical College. All study participants were informed about the study by a nurse or doctor fluent in Kiswahili and provided written informed consent before participation. All results were made available to clinicians and recorded in the patient’s file. Disease management was conducted by the health care workers of the clinic according to Bugando Medical Centre and Tanzanian management protocols and practices.During the study period, 488 adults were screened and 34 were excluded from the study: 7 patients were found to be pregnant (3 HIV-infected ART-naïve and 4 HIV-infected on ART), and 27 did not return the following day . A total of 454 adults were enrolled: 153 HIV-negative controls, 151 HIV-infected ART-naïve adults and 150 HIV-infected adults on ART.2 vs. 23.8 (22.3–25.8) and 23.7 (21.5–27.9)) and were severely immunosuppressed (mean CD4 T-cell count 215 (150–321) cells/μL vs. 378 (263–521) in the group on ART). HIV-infected adults on ART had been using ART for a median of 56 (31–68) months; 18 (12.0%) were taking protease inhibitors, which was a combination of lopinavir and ritonavir for all 18 of them.The characteristics of the 3 groups were broadly similar. Notable differences included that HIV-infected adults on ART were slightly older (median age 40 (38–47) vs. 38 (32–46) and 37 (32–44) years in the other 2 groups), more likely to be female (76.7% vs. 61.4% and 58.9%) and had a higher prevalence of central obesity (52.0% vs. 28.8% and 37.1%). HIV-infected, ART-naïve adults had lower mean BMI (22.0 (20.2–24.3) kg/mOverall, GMDs were more common among HIV-infected adults on ART . DM  and impaired glucose tolerance  were also more common among HIV-infected adults on ART. The prevalence of GMDs among HIV-infected, ART-naïve adults was similar to that of controls . Only one HIV-infected, ART-naïve adult had DM .Of the 30 total study subjects with IGT in our study, 11/30 had fasting blood glucose >6 mmol/L and therefore would have been labelled as IFG by the WHO definition used in this study if only fasting blood glucose measurements were obtained and OGTT had not been performed. Of these 30 study subjects with IGT, 26/30 had fasting blood glucose >5.6 mmol/L and therefore would have been labelled as IFG by the definition of the American Diabetes Association . The medThe number of subjects who reported prior glucose testing was <25% in all 3 groups: 33/153 (21.6%) in the control group, 16/151 (10.6%) in the HIV-infected, ART-naïve group and 18/150 (12.0%) in the HIV-infected, on ART group. Rates of awareness, treatment and control were low. Of 36 subjects with DM, only 2  in the HIV-infected, on ART group) were aware of their diagnosis. None of the subjects with DM were controlled according to the IDF definition .In the partially-adjusted logistic regression model of all factors listed in We also performed multiple, pre-determined multivariable logistic regressions to determine if the higher rates of GMDs among HIV-infected, on ART could explained by other confounders known to be associated both with HIV status and GMDs. The results of these models are shown in There was a strong and dose-dependent association between GMDs and grade of hypertension among HIV-negative adults but this association was not statistically significant among HIV-infected adults. See In our study in Tanzania, 1/3 of HIV-infected adults on ART for ≥2 years were found to have GMDs, a prevalence that was 4x higher than among HIV-negative study subjects and similar to HIV-infected adults in the US and Europe ,19, and + T-cell count was higher in both these groups of HIV-infected adults compared to our study. Study results from SSA have been mixed. Mercier and Ngatchou found that there was a higher prevalence of GMDs among adults who were HIV-infected, ART-naïve compared to HIV-negative controls [In our study, the prevalence of all GMDs among HIV-infected, ART-naïve adults was low, similar to the prevalence among HIV-negative controls. Data are few regarding the prevalence of GMDs among HIV-infected, ART-naïve adults in SSA. One study from Italy demonstrated IFG was more common among HIV-infected, ART-naïve adults compared to HIV-negative controls , and thecontrols ,26, but controls ,27,28. ODisturbingly, very few HIV-infected adults with diabetes mellitus were aware of their diagnosis. In fact, among HIV-infected adults on ART with diabetes mellitus, only one (<5%) knew that they had diabetes mellitus. Similarly low rates of DM awareness have been reported in other studies from Africa ,30, but The higher prevalence of GMDs among HIV-infected adults on ART was not explained by the most likely confounders such as difference in waist-hip ratio, BMI, age, gender and socioeconomic status. Even after adjusting for differences in age, gender, adiposity and socioeconomic status, HIV-infected adults on ART were still found to have a nearly 6-fold greater odds of having GMD compared to the HIV-negative control patients. The most likely alternative explanation is that GMDs may be a direct result of ART use since some, but not all , studiesThe higher prevalence of GMDs among HIV-infected adults on ART may be related to dysregulated inflammation in the setting of severe immunosuppression and subsequent immune reconstitution. In our study a higher current CD4 was associated with GMDs in both univariable and minimally adjusted analyses; HIV-infected adults with a current CD4 >500 had a 3-fold greater odds of GMDs compared to those with a CD4 <200, and a clear dose-response was seen. Our findings seem to fit with those recently reported from a prospective randomized ART-initiation trial in the United States and Puerto Rico, in which the development of higher fasting plasma glucose levels over a 96-week period on ART was significantly associated with higher baseline HIV-1 RNA levels . Other dOur study has limitations. Since this study is cross-sectional, we cannot make any conclusions as to causality. Nevertheless, this study does generate some interesting hypotheses as to the higher prevalence of GMDs among HIV-infected adults on ART. In addition, our HIV-negative control group may not be representative of the general population, although the prevalence of diabetes mellitus was similar to a recent representative population survey in our city. Further, haemoglobin A1C and HIV viral load testing are not currently available at our hospital. Also, additional data that may have been useful, such as family history of diabetes, hepatitis C status, and weight change from baseline, were not available for our study subjects.+ T-cell counts. Clearly, diabetes mellitus screening and management must be integrated into routine HIV care. In addition, further studies are needed to determine the pathophysiologic explanation for the higher prevalence of GMDs among HIV-infected adults on ARTs and whether this may be related to dysregulated inflammation in the setting of immune reconstitution.In conclusion, HIV-infected Tanzanian adults on ART had a 4-fold higher prevalence of GMDs than HIV-negative control patients. One-third of these adults had some GMD, 20% had diabetes mellitus and 15% had IGT. Among the 27 with diabetes mellitus who had completed at least two years of ART, only one was aware of their diagnosis and the remaining 26 were asymptomatic. In contrast, the prevalence of GMDs among HIV-infected, ART-naïve adults was low, similar to HIV-uninfected adults, and the only subject with diabetes mellitus was already diagnosed. The higher prevalence of GMDs among HIV-infected adults on ART could not be explained by differences in adiposity, age, gender or socioeconomic status but were associated with CD4


Previous studies have identified the beneficial effects of electroacupuncture (EA) on motor behaviors in Parkinson's disease (PD). However, the role and potential mechanisms of EA in PD-associated depression remain unclear. In the present study, a rat model of PD with unilateral 6-hydroxydopamine (6-OHDA) lesions in the medial forebrain bundle was treated using EA for 4 weeks. We found that 100 Hz EA improved several motor phenotypes. In addition, tyrosine hydroxylase (TH) immunohistochemical analysis showed that EA had a minimal impact on the TH-positive profiles of the ipsilateral ventral tegmental area. Compared with the 6-OHDA group, long-term EA stimulation significantly increased sucrose solution consumption and decreased immobility time in the forced swim test. EA treatment did not alter dopamine, norepinephrine, and serotonin levels in the striatum and hippocampus. Noticeably, EA treatment reversed the 6-OHDA-induced abnormal expression of brain-derived neurotrophic factor (BDNF) and tropomyosin-related kinase B (TrkB) in the midbrain and hippocampus. These results demonstrate that EA at 100-Hz possesses the ability to improve depressive-like symptoms in PD rats, which is, at least in part, due to the distinct effect of EA on the mesostriatal and mesocorticolimbic dopaminergic pathways. Moreover, BDNF seems to participate in the effect of EA in PD. Parkinson's disease (PD) is generally recognized as a progressive neurodegenerative disorder characterized by motor dysfunction. However, accumulating evidence indicates that nonmotor symptoms of PD, such as sensory dysfunction, sleep disturbance, and psychiatric complications , are more detrimental to well-being and functional ability than motor symptoms –3. As onAcupuncture, a traditional complementary and alternative medical approach, has been widely used as an adjunct to standard therapy for PD. Although the definite role of acupuncture in PD is controversial , increasBrain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, plays critical roles in cell differentiation, neuronal survival, migration, and synaptic plasticity. BDNF is reportedly involved in the pathophysiology of both PD and depression, as evidenced by the fact that altered expression of BDNF mRNA and protein has been observed in postmortem studies of PD patients . MeanwhiAnimal models are an important way to improve our understanding of the pathophysiological mechanisms of PD and possible treatments. Studies have suggested that bilateral infusion of 6-hydroxydopamine (6-OHDA) into the striatum , SNc 2525, 26, o ad libitum. All experimental procedures were performed according to the Ethics Committee on Animal Care and Usage of Capital Medical University.Adult Sprague-Dawley (SD) rats weighing 200–220 g were obtained from the animal facility of Capital Medical University. Rats were housed three per cage under standard laboratory conditions: 12-h light/dark cycle, 22°C room temperature, 55% relative humidity, and access to food and waterμg  6-OHDA (Sigma-Aldrich) was administered, at a rate of 0.5 μL/min. The microsyringe was left in place for 4 min to allow diffusion. Sham-operated rats underwent the same surgical procedure but received saline injection. Behaviors were assessed 2 weeks after surgery.Rats were anesthetized with 2% pentobarbital sodium  and fixed into a Kopf stereotaxic apparatus. Lesions were induced by injection of 6-OHDA using a 30-gauge needle Hamilton syringe into the right MFB at the following coordinates: anteroposterior (AP), –4.3 mm; mediolateral (ML), –1.5 mm; and dorsoventral (DV), 7.6 mm from the dura. A total dose of 8 EA stimulation was administered from 2 weeks to 6 weeks after 6-OHDA lesions were induced in the rats. Rats were randomly divided into 4 groups: a vehicle-treated sham group, a 6-OHDA-lesioned model group, and two groups of 6-OHDA-lesioned rats receiving EA at either 0 or 100 Hz. Two stainless steel needles of diameter 0.25 mm were inserted at a depth of 5 mm into the acupoints of BAIHUI  and DAZHUI . A bidirectional square wave (0.2 ms) electrical pulse with frequency 100 Hz was administered from a medical EA apparatus  for 30 min a day, 6 days a week for 4 weeks. The intensity of stimulation was increased stepwise from 1 to 2 mA and then to 3 mA, with each step lasting for 10 min. During EA stimulation, the rats were kept in the cage in an awake, unrestrained condition. Those treated with EA at 0 Hz underwent the same procedures, but no electrical pulses were delivered through the needles.The rotarod test was used to evaluate balance and motor coordination. The initial 3 days of testing served as training, and rats were placed on the rod turning at a low speed, permitting them to attain stable performance. On the test day, an accelerating protocol going from 4 rpm to 40 rpm in 2 min was performed, and the time taken for the rats to fall was automatically recorded.To assess general locomotor activity and anxiety-related activity, the rats were monitored using automatic infrared beams in a black chamber . The rats were placed in the arena and allowed to freely move about for 30 min while being recorded by an overhead camera. The footage was then analyzed using DigiScan analyzer and software  to extract the following parameters: horizontal movement distance, vertical movement distance, and numbers of entries into the arena center.This behavioral test was performed in a blinded fashion. The rats received a subcutaneous injection of 0.05 mg/kg apomorphine hydrochloride (Sigma) dissolved in 1% ascorbic acid and 0.9% NaCl. Immediately after apomorphine injection, the net number of rotations was recorded and assessed over 30 min. Rotational testing was performed in automatic rotameter bowls .The sucrose preference test is used as an indicator of anhedonic behavior in rodents. Before the beginning of testing, rats were kept individually habituated to two needleless syringes  8 h per day for 2 days in their home cage. After training, the rats were deprived of food and water for 12 h and then exposed to the two syringes for 30 min as described previously . After aThe forced swim test (FST) is commonly used to assess behavioral despair in rodents , 23. Theμm thick slices on a cryostat and processed for TH immunohistochemical analysis. Brain slices were incubated overnight at 4°C with TH antibody , followed by incubation with a biotinylated secondary antibody  for 30 min. The antibody was detected with an avidine/biotine complex  and visualized with 3,3-diaminobenzidine (Sigma). Six identical TH-labeled slices spanning the entire mesencephalon and including both SNc and VTA brain regions were selected from 5 animals per group. Using a bright-field microscope , the boundaries of the SNc and VTA areas were drawn. The number of TH-immunoreactive profiles in these areas was counted by stereology, using Stereo Investigator software  as described previously [Rats were sacrificed and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer. Frozen mesencephalon coronal sections were cut into 30-eviously . The surμL) was mixed with 80 μL mobile phase solution and was kept in the dark in an ice-bath (60 min), followed by centrifugation . The resultant supernatant was injected into the chromatograph. The mobile phase solution  comprised 63.5 mM citric acid monohydrate, 60.9 mM trisodium citrate dihydrate, 0.1 mM EDTA, 0.5 mM 1-octanesulfonic acid sodium salt, and 8% methanol. The pH of the running buffer solution was adjusted to 4.0, and it was then filtered through a filter of pore size 0.45 mm. The peak areas of external standards were used to quantify sample peaks.Rats were sacrificed by decapitation 1 day after behavioral measurements. The striatum, ventral midbrain, and hippocampus were rapidly dissected and stored in liquid nitrogen at –80°C. The concentrations of DA, 5-HT, and NE were assayed using reverse-phase high-performance liquid chromatography (HPLC) with electrochemical detection. The tissue samples were homogenized with an ultrasonic cell disrupter (Sonics) in 0.4 M perchloric acid. After centrifugation at 10,000 ×g for 30 min, the supernatant (160 The level of BDNF was determined by enzyme-linked immunosorbent assay (ELISA). The ventral midbrain and hippocampus were rapidly dissected and homogenized in a lysis buffer containing protease inhibitors (Sigma-Aldrich). The sample protein contents were measured using a BCA protein assay kit . For ELISA, a BDNF ELISA kit was used according to the manufacturer's instructions . BDNF levels were determined relative to a standard curve constructed using BDNF protein standards that were assayed simultaneously with the experimental samples.The expression of TrkB was determined by western blotting. The ventral midbrain and hippocampus tissues of rats were prepared as described previously . Equival post hoc test for comparison of groups. P < 0.05 was the criterion for statistical significance.Data were reported as means ± SEM. One- or two-way analysis of variance (ANOVA) was used, as appropriate, followed by the Newman-KeulsP < 0.05), while 100-Hz EA treatment, but not 0-Hz EA treatment, reduced the intensity of apomorphine-induced turning. Sham animals (n = 19) showed no turning behavior. Motor coordination was assessed using the rotarod test  and weeks of treatment  and an interaction of group × weeks . Further post hoc analyses showed that 6-OHDA-lesioned rats developed significant coordination deficits  before EA treatment. However, 100-Hz EA did not reverse this latency after 1 (P > 0.05) or 2 weeks of treatment (P > 0.05) but showed some effect following 3 (P < 0.001) and 4 weeks (P < 0.001) of treatment compared with the 6-OHDA model group. Meanwhile, an obvious decrease in the horizontal movement distances was found in the 6-OHDA group compared to the sham group  before EA treatment. However, 100-Hz EA significantly increased horizontal movement distances after 3 (P < 0.001) and 4 weeks (P < 0.001) of treatment. Similar to previous reports, 0-Hz EA showed no effect on these motor deficits [The apomorphine-induced turning test was performed 6 weeks after 6-OHDA lesions were induced in the rats. As shown in rod test . A repeadeficits . These dP < 0.001, Figures P > 0.05, Figures P > 0.05). These results collectively support the contention that 100-Hz EA can alleviate the abnormal movement symptoms triggered by 6-OHDA lesion and has a minimal impact on the mesocorticolimbic dopaminergic pathway but not on the mesostriatal pathway.Immunohistochemical staining for TH was performed 6 weeks after the MFB lesions were induced, and the numbers of TH-immunoreactive neurons in the SNc and VTA were quantified. The data show that 6-OHDA MFB lesions induced loss of TH-positive profiles by 94.5% ± 1.8% in the SNc on the ipsilateral side compared with the unlesioned side  and week  but no significant effect of the group × week interaction . Bonferroni post hoc analysis shows that all 6-OHDA-lesioned rats displayed a significant decrease in sucrose solution intake 2 weeks after neurotoxin exposure compared to the sham rats  and 4 weeks (P < 0.05) of EA treatment compared with the 6-OHDA group.Depressive-like behavioral phenotypes were assessed after 4 weeks of EA treatment. Anhedonic behavior was measured using the sucrose preference test. Two-way ANOVA showed significant effects of group (P < 0.05). However, 4 weeks of EA treatment effectively reduced immobility time compared to 6-OHDA group (P < 0.05). Analysis of swimming and climbing behavior in the FST demonstrated that 6-OHDA MFB lesions significantly affected these parameters . Similarly, EA treatment effectively increased climbing time compared to 6-OHDA group (P < 0.05).FSTs were performed to evaluate behavioral despair in depressive rats. Six weeks after neurotoxin exposure, the 6-OHDA group exhibited a significant increase in immobility time compared with the sham group  but no significant effects of week  and interaction between group and week   and 4 weeks (P < 0.05) of treatment. Thus, continuous EA stimulation has a strong positive effect on reversing the anhedonic-like and despair-like behavioral phenotypes in 6-OHDA-lesioned rats.Moreover, the open field test was used to assess anxiety-related behavior. Two-way ANOVA showed significant effects of group (= 0.453) . In the P < 0.001). Similarly, the DA has a tendencious but not significant decrease in the lesioned ipsilateral hippocampus compared with the level in the sham group  and in the hippocampus (P < 0.001). Additionally, the 5-HT and NE levels in the striatum were unaffected by 4 weeks of treatment with 100-Hz EA  on the lesioned side of ventral midbrain and a decreased BDNF level (P < 0.05) on the lesioned side of hippocampus compared with the sham group, respectively. However, these alterations in BDNF levels on the lesioned side induced by 6-OHDA were reversed by EA treatment both in the ventral midbrain and in the hippocampus. Although the changes in BDNF level on the unlesioned side were modulated by EA treatment in the ventral midbrain, it has no influence on the unlesioned side in the hippocampus . 100 Hz EA treatment could increase TrkB expression on the lesioned side both in the ventral midbrain and in the hippocampus  occur, there are more than 50% of dopaminergic neurons that are lost in the SNc, exceeding 60–80% DA depletion in the striatum –37. In tPrevious studies indicated that the 6-OHDA MFB lesion model is a relevant tool to study the pathophysiology of both the motor and nonmotor symptoms of PD, including severe motor dysfunction and depressive-like symptoms . These bIn the present study, we demonstrated that high-frequency EA improves the motor symptoms as well as the nonmotor symptoms in PD rats. Similar to our previous studies , 15, thiAlthough PD is widely known to be a disorder associated with the depletion of striatal DA, prior evidence has indicated that dysfunctions of other neurotransmitters, such as NE and 5-HT, also participate in the development of depression in PD . AccordiClinical evidence has also shown that 5-HT alterations occur in patients with PD-associated depression . The admIn the current study, we found that 6-OHDA induced a marked reduction in the levels of DA, 5-HT, and NE in both the striatum and hippocampus, which suggests that dysfunctions in these neurotransmitters may contribute to many nonmotor symptoms of PD. Thus, correlations may exist between swimming in the FST and the serotonergic system or between immobility in the FST and the dopaminergic system. Therefore, one or more of these neurotransmitter systems may play an important role in depressive-like behavior in this PD model, and interventions aimed at restoring monoamine function may be beneficial in treating the disease , 36. HowAs a key regulator of synaptic development and plasticity, BDNF has been recognized to play a pivotal role in several neurodegenerative and psychiatric disorders . Our preCorrelations between depressive-like symptoms and the reduction in BDNF mRNA and protein levels in the hippocampus have been identified in postmortem evidence , 17. Fur2A receptors to alter serotonin transmission [In contrast, 6-OHDA lesions induced an increase in BDNF expression in the midbrain. There are some complex reasons as to why this may be. Because BDNF is highly expressed by dopaminergic neurons, the degeneration of dopaminergic neurons should induce decreased BDNF expression. However, it has been previously shown that 6-OHDA injection triggers microglia and astrocyte proliferation and increases BDNF-immunoreactivity in the lesioned midbrain , 49. In smission . Therefosmission . This diIn the present study, alterations in the BDNF levels and TrkB expression induced by 6-OHDA lesions were reversed by EA treatment in the hippocampus and midbrain. Therefore, we concluded that EA might modulate BDNF-TrkB interaction in the mesolimbic dopaminergic pathway. Moreover, BDNF seems to participate in the effects of EA and may serve as a potential therapeutic target in PD.In summary, we demonstrated that high-frequency EA improves the motor symptoms as well as the nonmotor symptoms in PD rats. The effects of EA in improving depressive-like symptoms might be partially attributed to the neuroprotective role of EA in the mesocorticolimbic dopaminergic pathways. 100-Hz EA did not elicit any significant changes in the level of 5-HT and NE either in the striatum or in the hippocampus of the 6-OHDA-lesioned rats. Interestingly, BDNF-TrkB pathway seems to participate in the effect of EA and may serve as potential therapeutic targets in PD.